The N-terminus of Himar1 mariner transposase mediates multiple activities during transposition

Abstract

Mariner family transposons are perhaps the most widespread transposable elements of eukaryotes. While we are beginning to understand the precise mechanism of transposition of these elements, the structure of their transposases are still poorly understood. We undertook an extensive mutagenesis of the N-terminal third of the transposase of the Himar1 mariner transposon to begin the process of determining the structure and evolution of mariner transposases. N and C-terminal deletion analyses localized the DNA binding domain of Himar1 transposase to the first 115 amino acids. Alanine scanning of 23 selected sites within this region uncovered mutations that not only affected DNA binding but DNA cleavage as well. The behavior of other mutations strongly suggested that the N-terminus is also involved in multimerization of the transposase on a single inverted terminal repeat and in paired ends complex formation which brings together the two ends of the transposon. Finally, two hyperactive mutations at conserved sites suggest that mariner transposases are under a pattern of stabilizing selection in nature with regard to how efficiently they mediate transposition, resulting in a population of "average" transposons.

Mariner Mos1 transposase optimization by rational mutagenesis

“…The corresponding HIMAR1 positions are indicated. Mutant activity was described by Lampe et al (1999), and Butler et al (2006) for HIMAR1, Pledger and Coates (2005) and this study for MOS1 Genetica (2009) 137:265-276 267 of the five selected was able to carry out transposition events with intact transposon ends, and TA duplication at the insertion sites. For each of the MOS1 mutants, five independent and newly-inserted transposons were therefore cloned and sequenced (data not shown).…”

“…Based on the data described above, the simplest hypothesis suggests that when expressed in mammalian cells, MOS1 undergoes phosphorylation at at least one of these positions, thus inhibiting its activity as a result of its reduced ability to bind to the ITR. This hypothesis is supported by the fact that HIMAR1 (which is active in human cells) had no phosphorylable residues at positions corresponding to two of these positions: T88 and S104 in MOS1, which correspond to K87 and G103, respectively in HIMAR1 (Butler et al 2006). The third residue, S99, which corresponds to S98 in HIMAR1, is predicted to be non-phosphorylated.…”

“…Five single-point mutations allocating transposition hyperactivity to HIMAR1 have been reported in various studies (Lampe et al 1999;Butler et al 2006;Keravala et al 2006) (Fig. 1).…”

“…Before analyzing mutant combinations, we checked whether each -Gouillou et al 2005a, b;Butler et al 2006), the HTH motif involved in ITR binding in blue (Brillet et al 2007), and the catalytic domain (DD34D) in pink (Richardson et al 2006). Single mutations used to obtain hyperactive MOS1 are indicated.…”

“…This required producing and purifying MOS1[? ], FET and FETY using fusions with MBP (Maltose Binding Protein), as this made it possible to purify active mariner transposases without carrying out a denaturing step (Auge-Gouillou et al 2005a, b;Butler et al 2006). FET and FETY were therefore cloned in the pMal plasmid.…”